Arabidopsis thaliana ecotype Columbia (Col-0), which was used as the wild-type control in the present study, was kindly provided by Dr. Yangdou Wei at the University of Saskatchewan, and the mutants fls2, bik1, bak1-4, bik1 sid2 were kindly donated by Dr. Libo Shan (Texas A & M university). sid2 was bought from the Arabidopsis Biological Resource Center. The bik1 mutant line was further identified as a homozygous mutant. The RT-PCR results showed that the bik1 mRNA could not be detected in homozygous mutant plants (Figure S1), suggesting that these plants represent knockout mutants at the BIK1 locus. The bik1 sid2 double mutant line was identified as a homozygous mutant. The RT-PCR results showed that the bik1 and sid2 mRNA could not be detected in homozygous mutant plants (Figure S2).

P. brassicae strain ZJ-1 was originally isolated from a diseased plant in a rapeseed field in Zhijiang County, Hubei Province, P R China. The virulence of single-spore isolated from P. brassicae strain ZJ-1 was tested on the differential hosts of Williams, showing the single-spore isolate derived from race 1 (Williams, 1966; Fähling et al., 2004). Resting spores of P. brassicae were extracted from clubroot galls (Asano et al., 1999), surface disinfested by freshly prepared 2% chloramine-T solution at room temperature for 20 min, washed twice with sterile water, adjusted to a concentration of 1.0 × 10⁷ spores per mL, and were then stored at 4°C. P. brassicae was proliferated using the stored resting spores in greenhouse with rapeseed. Arabidopsis Col-0 and mutant seeds were germinated on the surface of vermiculite in small pots. The seedlings were transplanted to the soil at 14 days post-germination. After growing of 7 days in a growth chamber, plants were inoculated with 1 mL of the resting spore suspension (1 × 10⁷ spores per mL) by injecting the soil around each plant. Arabidopsis and mutant plants grown in the 50 holes plate (54 cm × 28 cm × 5 cm), each seeding planted in one hole, the wild type and mutants Arabidopsis were planted in the same holes plate to reduce errors. The plants were grown in a plant growth chamber maintained at 70% humidity and 23°C with a 16/8-h day/night cycle. For all the mutants analyzed, gene expression was verified at 21 or 28 days post inoculation. Disease severity was assessed using a scoring system of 0–4 modified from Siemens reported (Siemens et al., 2002). A score of 0 indicated no disease; 1, very small galls mainly on lateral roots that did not impair the main root; 2, small galls covering the main root and few lateral roots; 3, medium to large galls, also on the main root; and 4, severe galls on lateral root, main root or rosette, with fine roots completely destroyed. Disease index (DI) was calculated using the five-grade scale according to the formula: DI = (1n₁ + 2n₂ + 3n₃ + 4n₄) × 100/4N_t, where n₁–n₄ is the number of plants in the indicated class and N_t is the total number of plants tested.

Reactive oxygen species (ROS) production was detected using the nitroblue tetrazolium (NBT) staining method (Montiel et al., 2012; Arthikala et al., 2014). The plants grown in the soil pot for 21 days after infection were used to determine O2- concentrations. The healthy roots and the galled roots were incubated for 5 h in dark at room temperature, and the roots were cleared in 90% ethanol.

To estimate the produced H₂O₂ in situ, the roots were hand-sectioned using a double-edged razorblade. The sections were subsequently rapidly immersed in 1 mg/ml of 3′, 3′-diaminobenzidine (DAB) solution, vacuum infiltrated for 2 min, and incubated for 2–3 h at 25°C in darkness (Gorska-Czekaj and Borucki, 2013).

A stock solution of SA (99.5%, Sigma Aldrich, dried substance) was prepared in ethanol/ddH₂O (v/v1:1), and diluted with sterile water at 2.5 × 10⁻⁵mol/L concentration for spraying (Lovelock et al., 2013). MeSA (99%, VETEC) was diluted in 10% ethanol and was sprayed at the 2.5 × 10⁻⁵mol/L concentration. SA and MeSA were used for three times of exogenous treatments. The first treatment was conducted 2 days prior to the inoculation, while the second treatment was carried out 2 days after inoculation, and the last was performed 10 days after inoculation. The total volume of each spraying was 2 L, and the remaining water was gently poured into the holding tray for slow absorption through the roots. Approximately 30 plants were treated for each sample (Lovelock et al., 2013).

DNA was extracted from root samples using the cetyl trimethyl ammonium bromide (CTAB) method (Allen et al., 2006). Quantitative PCR was performed on a CFX96 real-time PCR system (BioRad) using iTaq Universal SYBR Green supermix (BioRad) to quantify the P. brassicae target actin gene AY452179.1. Each reaction was performed with 2.5 ng of total DNA as template, and Arabidopsis actin gene AT3G18780 was used as an internal control for data normalization. Standard curves were constructed using serial dilutions of DNA extracted from the roots of Col-0 at 21 days after inoculation with P. brassicae, which was defined as a reference condition. Quantitative results were then expressed as the % of the P. brassicae mean DNA content in this reference condition (Lemarié et al., 2015a,b).

Total RNA was isolated from control roots, and the roots were inoculated with P. brassicae using TRIZOL reagent (Invitrogen). RNA samples were treated with DNase I to remove potential contaminating genomic DNA, followed by extraction with phenol:chloroform. First-strand cDNA was prepared using oligo (dT) primer. Quantitative PCR was performed on a CFX96 real-time PCR system (BioRad), using iTaq Universal SYBR Green supermix (BioRad). The following cycling conditions were used: 95°C for 30 s, 95°C for 5 s, 60°C for 15 s, and 72°C for 12 s. The reaction was performed for 40 cycles, followed by a step at 72°C for 5 s. Each amplification used 3 technical replicates the results of which were averaged to give the value for a single biological replicate. The primer sequences are provided in Table S1. We tried using the two Arabiodopsis genes (actin and ubiquitin) for qRTPCR, and the results were similar. The data shown in this paper selected Arabidopsis ubiquitin10 (AT4G05320) served as an internal control for normalization.

Fluorescent and transmission electron microscopy (TEM) were performed using the following protocol. For fluorescent microscopy, the healthy roots or galled roots were treated with Nile red solution (10 μg/ml Nile red in acetone) for 3–5 s, and excess dye was removed after brief rinsing in H₂O. The root samples were subsequently observed using Nikon fluorescence microscopy. Nile red emits fluorescence over a broad range of wavelengths, but observation using a filter set for B excitation (used for FITC, Cy2, Alexa488, or GFP) generates the best images (Suzuki et al., 2013). For electron microscopy analysis, the roots were fixed in 2.5% glutaraldehyde for 4 h, and were subsequently postfixed in 1% osmium tetroxide for 3 h, washed, dehydrated through an ethanol series, and embedded in London resin white. Ultrathin sections were examined through TEM (HITACHI, H-7000).

Wild-type and mutant Arabidopsis were infected with P. brassicae for 21 days, and uninfected roots were used as the control. The healthy root tissues and galls were carefully washed with tap water and embedded in a frozen embedding medium (Sakura Finetek USA, Inc., Torrance, CA) at −23°C overnight. Transverse root sections (50 μm thick) were sliced using a freezing microtome (Leica CM1950, Germany) and attached to the slides. A drop of water was added, and the specimens were covered with cover glass. The plant cells and spores were observed using a Nikon light microscope.

Statistical analysis was performed using SPSS (13.0) and a statistics package (Microsoft Excel 2010). In all experiments, One-way ANOVA, specifically Tukey's test with a P = 0.05, was used to analyze significant differences between treatment groups.

The symptoms of P. brassicae-infected plants included the formation of club-shaped galls on the roots, and the wilting, yellowing and premature senescence of the shoots. The plant defense response upon P. brassicae infection is often reflected by reduced gall size, root condition, and reduced resting spore production. To determine whether several known receptor-like kinases have function in clubroot-associated defense responses, we observed the infection and colonization of P. brassicae on the roots of the loss-of-function mutants (Figure 1). The roots of wild-type Arabidopsis infected by P. brassicae were formed of a typical galled, resulting in few rootlets; besides, the infected plants were yellowing (Figure 1A and Figure S3), and the ratio of gall formation was 100% (Figure 1B). The symptoms of fls2 and bak1 infected by clubroot-pathogen were similar to those of the wild type (Figure 1A and Figure S3), with a gall formation ratio of nearly 100% (Figure 1B). Interestingly, in bik1, the gall formation was inhibited as 79% of the plants that did not form a gall, and the root systems of P. brassicae-inoculated bik1 plants still developed with plentiful lateral roots (Figures 1A,B). The bik1 mutant line was identified as a homozygous mutant, and the RT-PCR results showed that the bik1 mRNA could not be detected in homozygous mutant plants (Figure S1), suggesting that these plants were the knockout mutants at the BIK1 locus. To evaluate P. brassicae production in the galls, the actin gene expression levels of P. brassicae were measured using quantitative real-time PCR. For the phenotype of no gall formation on bik1-infected roots due to the largely reduced P. brassicae production (by 99.78%), and for the phenotype of mid gall formation, root spore production was reduced by approximately 92% (Figure 1C). The relative amount of P. brassicae DNA in total root-extracted DNA was evaluated through quantitative PCR (Figure 1D). The results indicated that root pathogen density was not reduced within the infected roots of fls2 and bak1 mutants compared with Col-0, suggesting that fls2 and bak1 mutants had no significant change of pathogen density within the root samples. However, the density of P. brassicae in the infected roots with and without the formation of small galls in bik1 mutant was approximately 40 and 90% less than that in Col-0, respectively (Figure 1D). Taken together, the results showed that bik1 plants exhibited strong resistance to P. brassicae.

To observe the P. brassicae development state in the galls of the infected roots, longitudinal sections of Arabidopsis control and loss-of-function mutant roots were observed. There was no zoospores in the negative control (uninfected wild type Arabidopsis root cells), and many resting spores were found in infected wild type, fls2 and bak1 root cells. No obvious zoospores were observed on the infected roots of bik1, which showed no gall formation phenotype (Figure 2A). TEM was used to clearly demonstrate the zoospore developmental stages in the very small galls on bik1 root. The development of P. brassicae was in the state of multinucleate secondary plasmodium during cell division on bik1 root (Figure 2Bb), while the wild-type root cells were filled with resting spores and the cell division was completed (Figure 2Ba). These observations suggested that bik1 had inhibited development of P. brassicae, which explains the results that the wild-type roots showed severe galls with a high production of resting spores, while the bik1 roots had fewer and smaller galls with less P. brassicae.

The life cycle of P. brassicae consists of three stages: survival in soil, root hair infection, and cortical infection (Schwelm et al., 2015). To investigate the potential involvement of BIK1 in the infection process, we compared the root hair and cortical infections between the wild-type and bik1 mutant plants, including the colonization rate of primary plasmodia, zoosporangia, and secondary plasmodia (Figures 3A,B). Nile red was used to label P. brassicae for convenient and rapid detection of the stages of infection. The roots of more than 15 plants were selected for the control and bik1 mutants and sliced into 1–2 cm segments. A total of approximately 100 root segments per sample were observed and counted to determine the presence of infection. Compared with root hair infection of the control (100% primary plasmodia and 87.17% zoosporangia, respectively), the root hair infection ratio of bik1 mutant roots was reduced, showing 40.40% primary plasmodia and 54.47% zoosporangia, respectively (Figure 3B). Compared with the control roots (85.70% secondary plasmodia), the bik1 mutant roots showed a reduced cortical infections ratio (43.17% secondary plasmodia) (Figure 3B). Thus, root hair and cortical infections appeared to be impaired by the knockout of BIK1 expression in the mutant.

ROS plays dual roles in plants, both as toxic compounds and as the key regulators of many biological processes, such as growth, cell cycle, programmed cell death, hormone signaling, development, stress, and defense responses (Mittler et al., 2004). Pathogen infection may elicit ROS production. We used nitroblue tetrazolium (NBT), a reagent that allows the visualization of superoxide accumulation, to evaluate the ROS production in P. brassicae-infected roots of wild type Arabidopsis, fls2, bak1, and bik1 mutants. NBT-formazan precipitates within the roots of all the mutants and control wild type were localized to the places of gall formation (Figure 4), and the NBT-formazan precipitates were much less abundant in the infected roots of bik1 than in the roots of wild type or fls2 and bak1 mutant plants (Figure 4). We further observed the galls of P. brassicae-infected Brassica rapa through 3-3′-diaminobenzidine (DAB) staining. The results showed that the galled roots had much higher H₂O₂ accumulation than healthy roots in the cortex, which was filled with millions of P. brassicae resting spores (Figure S4). These results indicated that ROS might be an indicator of defense responses.

SA is a central component of defense in plants against a number of biotrophic pathogens (Catinot et al., 2008; Vlot et al., 2009). Thus, we investigated the potential of SA to stimulate defense in Arabidopsis against P. brassicae, and methyl salicylate (MeSA) was used as the control. The formation of root galls was assessed at 3 weeks after three repeated treatments with SA or MeSA. SA treatment significantly reduced gall formation, and the proportion of galling was decreased by 90% (from 100 to 10%). MeSA treatment resulted in no reduction of gall formation (from 100 to 100%) (Figures 5A–C). The disease symptoms associated with P. brassicae were observed to be decreased in the roots treated with SA, but no observable change was found in shoot morphology (Figure 5D). Nile red staining clearly showed that a very small amount of spores were observed in SA-treated roots (Figures 5E–G). These results indicate that SA enhances the resistance against P. brassicae.

P. brassicae-induced plant defense pathways are often regulated through certain plant hormones, JA pathway, and SA pathway (Lemarié et al., 2015b). The loss of BIK1 function resulted in higher basal SA levels and PR1 gene expression than wild-type Col-0 Arabidopsis (Veronese et al., 2006; Lei et al., 2014). To determine whether the resistance to P. brassicae was conferred through the loss of BIK1 function involving defense-related plant hormones, we detected the expression levels of the SA-signaling marker gene PR1 and the ET/JA marker genes ERF1 and PDF1.2 at 21 days after infection of P. brassicae (Figure 6). In wild-type roots, the expression levels of PR1, PDF1.2, and ERF1 were 6.5, 9.4, and 2.2-folds up-regulated after infection, respectively. Similar results were obtained for fls2 mutant plants. However, the transcript level of the SA-responsive gene PR1 in bik1 mutant roots was 48.4-folds higher before P. brassicae infection and 11.5-folds higher after the infection compared with that in the wild type roots. These data implied that BIK1 might function as a negative regulator of SA accumulation both in the presence and absence of P. brassicae infection.

The loss of SID2 function blocks SA biosynthesis (Wildermuth et al., 2001). To assess the role of SA in bik1 resistance to P. brassicae, bik1, sid2, and bik1 sid2 double mutant plants were used for infection (Figure 7). The non-inoculated sid2 mutant did not show any observable change in morphology compared with the wild type (Figures 7A,D, Figure S5). Besides, the severity of clubroot symptoms induced by P. brassicae was similar to that of wild-type plants: both of them showed yellowing leaves, severely galled and rotten taproots, and similar density of P. brassicae were detected (Figures 7A–C,E). These results are consistent with those reported previously (Lovelock et al., 2016). Apparently, bik1 reduced the clubroot symptom and density of P. brassicae (Figures 7A–E). These results indicated that the elevation of SA accumulation was required for bik1 resistance. Surprisingly, in the bik1 sid2 double mutant with reduced SA accumulation (Laluk et al., 2011; Lei et al., 2014), the phenotypes were similar to those of bik1 plants. These plants showed leaves with serrated margins and wrinkled surfaces, occasional curling, partial recovery to wild type, and strong resistance to P. brassicae similarly to bik1 (Figure 7 and Figure S5). The bik1 sid2 double mutant line was identified as a homozygous mutant (Figure S2), which showed partial resistance to clubroot possibly due to an epistatic effect or some other unknown signal pathways that contribute to the resistance of bik1 against P. brassicae.

SA and the SA-dependent signaling pathways play a major role in modulating SAR. In Arabidopsis plants defective in SA accumulation, SAR is significantly impaired, and in plants where SA is either over-expressed or externally supplied, enhanced SAR is typically observed (Fu and Dong, 2013). The bik1 mutant plants showed higher SA level and up-regulated PR1 gene expression compared with Col-0, indicating that the bik1 mutant plants had enhanced SAR. To examine whether SAR is important for Arabidopsis to resist P. brassicae, the npr1-1 mutant was inoculated with P. brassicae. The results showed that at 21 days post inoculation, the symptoms of npr1-1 were more severe than those of Col-0 (Figure 8). The infected npr1-1 leaves turned yellow and showed root rot, while the control plants showed galls on the roots (Figures 8A–C). We then evaluated the disease index in infected Col-0 and npr1-1 plants (Figure 8D), and the index for Col-0 was 69.4, while that for npr1-1 was 88.1. These results indicated that npr1-1 was more susceptible to P. brassicae than Col-0. Thus, the SAR of Arabidopsis also contributed to the resistance against P. brassicae.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.